Project Summary: Myelin, the outgrowth of glial cells, tightly associates with axons and increases the speed of electrical impulses that travel along myelinated nerve fibers. The axo-glial interface at the paranode is the site for a critical series of adhesive junctions that contain the cell-adhesion molecules, Caspr and contactin on the axonal membrane and Neurofascin 155 (NF155) on the glial cell membrane. Notably, Caspr is aberrantly located in the axons from patients with the demyelinating human disease, multiple sclerosis (MS), suggesting its mislocalization may contribute to myelin loss and axonal degeneration. Therefore, detailed information about the structural organization of the axo-glial junctions will be useful for future studies related to the pathogenesis of demyelinating diseases. The broad, long-term research objective of the proposed studies is to use electron tomography to reveal the structural organization of axo-glial interactions, thus contributing to our current knowledge of the axon and glial cell architecture as well as the resulting cellular substructure from demyelinating diseases. The specific aims for the proposed studies are as follows: 1. Electron tomography will be conducted on myelinated axons to obtain high-resolution density maps of the axo-glial junctions. Sample sources of myelinated axons include wildtype mouse brain tissue and myelinating co-cultures, all of which will be prepared for electron microscopy using the sophisticated preparative techniques of high-pressure freezing and freeze substitution. Immunogold labeling and crystal- structure homology modeling of Caspr, contactin, and NF155 will be used in conjunction with the tomography to produce a model of the junctions that will contribute to our understanding of axo-glial interactions. 2. Experiments are planned to investigate the relationship between the axo-glial junctions and the axon cytoskeleton using electron tomography and the above mentioned techniques. 3. Finally, electron tomography will be conducted on myelinated axons from various knock-out mice for comparisons with the wild-type 3D structures of the junctions and other cellular components. I will use mice deficient for the junctional proteins and cytoskeletal proteins such as Protein 4.1B, spectrin, and neurofilament subunits. Relevance: This research seeks to determine the various structural differences between axons from normal mouse brains and axons from various demyelinating mouse models. Ultimately, this information will provide insight into the ultrastructure of axons from patients with multiple sclerosis and will provide valuable information for future research involving human neurological degenerative diseases.